title: Transcriptome dynamics of transgene amplification in Chinese hamster ovary cells.
abstract: Dihydrofolate reductase (DHFR) system is used to amplify the product gene to multiple copies in Chinese Hamster Ovary (CHO) cells for generating cell lines which produce the recombinant protein at high levels. The physiological changes accompanying the transformation of the non-protein secreting host cells to a high producing cell line is not well characterized. We performed transcriptome analysis on CHO cells undergoing the selection and amplification processes. A host CHO cell line was transfected with a vector containing genes encoding the mouse DHFR (mDHFR) and a recombinant human IgG (hIgG). Clones were isolated following selection and subcloned following amplification. Control cells were transfected with a control plasmid which did not have the hIgG genes. Although methotrexate (MTX) amplification increased the transcript level of the mDHFR gene significantly, its effect on both hIgG heavy and light chain genes was more modest. The subclones appeared to retain the transcriptome signatures of their parental clones, however, their productivity varied among those derived from the same clone. The transcript levels of hIgG transgenes of all subclones fall in a narrower range than the product titer, alluding to the role of many functional attributes, other than transgene transcript, on productivity. We cross examined functional class enrichment during selection and amplification as well as between high and low producers and discerned common features among them. We hypothesize that the role of amplification is not merely increasing transcript levels, but also enriching survivors which have developed the cellular machinery for secreting proteins, leading to an increased frequency of isolating high-producing clones. We put forward the possibility of assembling a hyper-productivity gene set through comparative transcriptome analysis of a wide range of samples.
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington Avenue S.E., Minneapolis, Minnesota, 55455-0132.
authors.forename: Nandita
authors.initials: N
authors.lastname: Vishwanathan
authors.forename: Huong
authors.initials: H
authors.lastname: Le
authors.forename: Nitya M
authors.initials: NM
authors.lastname: Jacob
authors.forename: Yung-Shyeng
authors.initials: YS
authors.lastname: Tsao
authors.forename: Sze-Wai
authors.initials: SW
authors.lastname: Ng
authors.forename: Bernard
authors.initials: B
authors.lastname: Loo
authors.forename: Zhong
authors.initials: Z
authors.lastname: Liu
authors.forename: Anne
authors.initials: A
authors.lastname: Kantardjieff
authors.forename: Wei-Shou
authors.initials: WS
authors.lastname: Hu
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics; D008727:Methotrexate / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012641:Selection, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D059467:Transcriptome; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: D004791:Enzyme Inhibitors; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate
keywords: Chinese Hamster Ovary (CHO) cells; dihydrofolate reductase (DHFR); gene amplification; hyper-productivity; mammalian cell culture; methotrexate (MTX); selection